Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.